Detection and quantitation of analytes in assays is important in immunology, microbiology, diagnostics and medicine. Some methods for detecting analytes rely on intrinsic properties of the molecule to be detected. For example, methods for detecting analytes in samples such as chemical analysis, include NMR, high pressure liquid chromatography, gas chromatography, absorbance, fluorescence, chemiluminescence and mass spectrometry. These techniques rely on chemical or physical properties of the molecules being detected (for example optical properties, reactive groups, charge, size, and hydrophobicity).
Other methods for detecting analytes rely on some part of the analyte being recognized by another molecule. For example, methods for detecting nucleic acids often utilize hybridization and complementary base pairing. Other methods rely on the conformation of part of the analyte being recognized by another molecule. Examples of such methods of detection include an antibody binding to an antigen and an aptamer binding to a target epitope region.
Examples of biological assays where these detection methods are utilized are innumerable, and include DNA sequencing, restriction fragment length polymorphism determination, Southern blotting and other forms of DNA hybridization analysis, determination of single-strand conformational polymorphisms, comparative genomic hybridization, mobility-shift DNA binding assays, protein gel electrophoresis, Northern blotting and other forms of RNA hybridization analysis, protein purification, chromatography, immunoprecipitation, protein sequence determination, Western blotting (protein immunoblotting), ELISA and other forms of antibody-based protein detection, isolation of biomolecules for use as antigens to produce antibodies, PCR, RT PCR, differential display of mRNA by PCR, and the like. Protocols for carrying out these and other forms of assays are readily available to those of skill in the art.
Methods that utilize recognition of an analyte by another molecule for detection are important, particularly in biology. One area where there is lack of available systems and useful reagents is in the area of near-infrared chemifluorescent systems. Chemifluorescent reactions occur very fast. However, it would be advantageous to be able to visualize a chemifluorescent reaction with the naked eye through a simultaneous chromogenic reaction. There is a need in the art for new chemifluorescent reagents that also have a chromogenic reaction associated with them. The present invention satisfies these and other needs.